Invasive pulmonary aspergillosis (IPA) due to A. fumigatus has emerged as a leading cause of mortality among immunosuppressed patients. The development of new therapeutic, vaccine and diagnostic strategies is limited by poor understanding of the pathogenesis of IPA. In an ongoing collaboration with Dr. Nguyen (Project I), we used In Vivo Induced Antigen Technology (IVIAT) to identify C. albicans proteins that are induced during oral thrush in HIV-infected patients, including novel virulence factors. In this project, we will adapt IVIAT to identify A. fumigatus antigens that are induced during the pathogenesis of IPA in humans. In the first specific aim, we will adsorb paired sera recovered from a patient: a) before the diagnosis, and b) after recovery from IPA against A. fumigatus antigens present in vitro. The pairs of adsorbed sera will then be used to screen an A. fumigatus genomic DNA expression library in parallel, identifying clones expressing immunogenic antigens. Antigens reactive with antibodies in the post-recovery serum but not reactive with antibodies in the pre-diagnosis serum are likely to be expressed during the pathogenesis of IPA. The paired sera will also be used to screen a cDNA expression library in parallel. In the second specific aim, the genes encoding the immunogenic antigens will be identified. In the third specific aim, we will purify recombinant antigens, raise antibodies and use them to localize antigens to A. fumigatus cells within the lungs of mice with IPA. Finally, in specific aim 4, we will compare serum antibody responses against in vivo induced antigens among: a) groups of patients with IPA and uninfected controls, and b) individual patients before the diagnosis and after recovery from IPA. We hypothesize that selected in vivo induced proteins are A. fumigatus virulence factors and, as such, might represent therapeutic, vaccine or diagnostic targets. These hypotheses will be tested in future studies, which will be similar to those proposed in the present Program Grant to characterize in vivo induced C. albicans proteins.